Alpha6* (alpha6-containing) nicotinic acetylcholine receptors (nAChRs) play an important role in nicotine addiction. Alpha6* nAChRs are found in just a few regions of the mammalian brain (the mesolimbic and nigrostriatal dopaminergic pathways) and mediate reward and motor control. Aim 1 develops and exploits a cell line expressing alpha6beta2beta3 nAChRs without the use of chimeric or concatameric subunits, which have both been required for the successful expression of this subtype in cell lines. Successful expression of alpha6beta2beta3 nAChRs will be aided by expressing two point mutations in the beta2 subunit, which have been found to enhance export from the endoplasmic reticulum (ER). In addition, a fluorescent marker for ER exit sites (Sec24D) will be used to identify cells with abundant ER exit sites which are a marker for high levels of alpha6beta2beta3 nAChR expression on the plasma membrane. Functional alpha6beta2beta3 will be characterized using electrophysiological techniques. A distinguishing factor that governs functional properties of nAChRs is the subunit stoichiometry of the assembled pentamers. Aim 2 therefore determines the subunit stoichiometry of alpha6beta2beta3 nAChRs using a novel technique involving zero-mode waveguides (ZMWs). ZMWs allow the single-molecule analysis of a single membrane receptor by seeding cells on nanofabricated apertures ~100 nm diameter. Combining fluorescent nAChR subunits and photobleaching techniques will allow the quantification of specific subunits in receptor assemblies. It is well known that some nAChRs (e.g., alpha4beta2) are upregulated when chronically stimulated by nicotine. This change in receptor number has been implicated as one of the mechanisms that is critical for nicotine addiction. To determine how alpha6* nAChRs may play a role in nicotine addiction, Aim 3 determines how these receptors are changed when exposed to nicotine by using mice that have fluorescent alpha6 nAChR subunits. The mice will be chronically exposed to nicotine and quantitative fluorescence will document the change in receptor number. The understanding of how nicotine, in comparison to other drugs (i.e., acetylcholine, epibatidine), modifies the functio and receptor levels of alpha6* nAChRs will provide novel insights regarding the mechanisms that mediate nicotine addiction. Characterization of alpha6beta2beta3 nAChRs, determination of alpha6beta2beta3 nAChR stoichiometry, and determination of upregulation of alpha6* nAChRs will advance the understanding of nicotine addiction and will further the development of novel smoking cessation therapeutics.